GlycoFi has engineered Pichia to produce recombinant glycoproteins with human-like glycosylation. However, the extensive genetic modifications have also caused fundamental changes in cell wall structures, predisposing these glyco-engineered strains to cell lysis and reduced cell robustness during fermentation. These undesirable traits have resulted in substantial reductions in cell viability as well as a marked increase in intracellular protease leakage into the fermentation broth, resulting in a reduction in both recombinant product yield and quality. Isolated fungal host cells, such as Candida albicans, Hansenula polymorpha; Schizosaccharomyces pombe; Saccharomyces cerevisiae; Pichia pastoris, lacking functional OCH1, a polypeptide in the fungal glycosylation pathway, are known to be temperature sensitive. For example, Candida albicans och1 knock-outs are temperature sensitive at 42° C. (Bates et al., Outer Chain N-Glycans Are Required for Cell Wall Integrity and Virulence of Candida albicans, The Journal of Biological Chemistry 281: 90-98 (2006); Hansenula polymorpha och1 knock-outs are temperature sensitive at 45° C. (Kim et al., Functional Characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 Genes as Members of the Yeast OCH1 Mannosyltransferase Family Involved in Protein Glycosylation, The Journal of Biological Chemistry, 281: 6261-6272 (2006)); Schizosaccharomyces pombe och1 knock-outs are temperature sensitive at 37° C. (Yoko-o et al., Schizosaccharomyces pombe och1+ encodes alpha-1,6-mannosyltransferase that is involved in outer chain elongation of N-linked oligosaccharides, FEBS Letters 489: 75-80 (2001)); Saccharomyces cerevisiae och1 knock-outs are temperature sensitive at 37° C. (Nakayama et al., OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides, EMBO J. 11(7):2511-9 (1992)); and Pichia pastoris och1 knock-outs are temperature sensitive at 37° C. (Choi et al., Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris, Proc Natl Acad Sci USA. 100(9):5022-7 (2003)). Additional genetic modifications to make och1− fungal host cells (e.g., Pichia cells) more robust in cell culture would be of value.
An unlikely candidate for genetic modification in order to increase Pichia culture robustness is CRZ1, a zinc finger transcription factor. CRZ1 is known to regulate a number of S. cerevisiae plasma membrane and cell wall regulatory genes (Cyert, Biochemical and Biophysical Research Communications 311:1143-1150 (2003)). Perturbation of plasma membrane and cell wall synthesis, due to mutation of CRZ1, would have been expected to make Pichia cells less robust. The published characterizations of S. cervisiae CRZ1 would have led a practitioner of ordinary skill in the art to predict that Pichia cells, lacking functional CRZ1, would be less viable and robust when placed under high temperature stress. (Matheos et al., Genes & Development 11:3445-3458 (1997); Stathopoulos et al., Genes & Development 11: 3432-3444 (1997)).